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276 12 Mining Genomes for Nitrilases

this subtype share high levels of similarity (over 90%) in bacteria (rhodococci), but
are less similar (with around 40% identity) to fungal nitrilases acting on aromatic
nitriles. This is, however, in accordance with the observation that the substrate
specificities of the fungal and bacterial enzymes are not quite the same, the former
also transforming some aliphatic nitriles.

12.3.2
Analysis of Specific Regions

With the aim of predicting mandelonitrile-hydrolyzing activities, a set of 174
nitrilase sequences (from cultured organisms, from metagenomes, hypothetical
proteins) was subjected to a sequential analysis of specific regions [9]. The ability to
accept bulky substrates such as mandelonitrile was postulated to be influenced by
five regions, those downstream of the Lys and Cys residues of the catalytic triade,
and three inserts. The positions of these regions in the enzyme subunit were
predicted using the known structure of the amidase from Pseudomonas aeruginosa
(member of the nitrilase superfamily).
According to these criteria, four enzymes were selected from the set and activity
for mandelonitrile was confirmed in two of them (from Burkholderia xenovorans and
Bradyrhizobium japonicum). However, the enzymes exhibited no enantioselectivity
for this substrate [9, 10]. Both of them carried an Ala residue in the direct
neighborhood of the catalytic Cys and the same residue was also found, for
instance, in the nitrilase from P. fluorescens [19] acting on mandelonitrile with a low
R-selectivity. However, some nitrilases carrying Trp at that position (for instance,
the enzymes from Alcaligenes sp., Pseudomonas putida, or a number of fungi [8, 23,
24]) also hydrolyzed mandelonitrile but with a high R-selectivity. The importance
of this aa residue for enantioselectivity was demonstrated by mutational studies
(see following text).
The region downstream of the catalytic Cys also allows nitrilases and cyanide
hydratases to be distinguished relatively successfully. The experimentally confirmed
cyanide hydratases contain an Asn residue at position 3 downstream of the catalytic
Cys [5, 6], whereas most of the characterized nitrilases carry a His at this position
except for a few enzymes (from A. thaliana and Klebsiella ozaenae with an Asn at
the corresponding site [3]).
12.3.3
Analysis of Enzyme Mutants

The site-directed mutagenesis of the nitrilase from P. fluorescens EBC191 (NitP)
focused on specific regions, first of all the C-terminus [25] and the vicinity of the
catalytic Cys [26, 27].
The C-terminal part of NitP was important for activity, amide formation, enantios-
electivity, and stability, as demonstrated by the examination of truncated variants,
which generally exhibited lower activities, increased amide formation, altered
enantioselectivity, and lowered resistance to freezing and thawing [25].